1. Field of the Invention
The present invention relates generally to compositions and methods useful for in vivo polynucleotide-based polypeptide delivery into cells of vertebrates. More particularly, the present invention provides the use of salts and/or auxiliary agents in compositions and methods useful for direct polynucleotide-based polypeptide delivery into the cells of vertebrates.
2. Related Art
The in vivo delivery of a polynucleotide (e.g., plasmid DNA) into vertebrate tissues has been shown to result in the cellular uptake and expression of the polynucleotide into a desired polypeptide (Wolff, J. A. et al., Science 247:1465-1468 (1990); Wheeler, C. J. et al., Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996)). Potential human therapeutic uses of such polynucleotide-based polypeptide delivery include immune response induction and modulation, therapeutic polypeptide delivery, and amelioration of genetic defects. For example, a polynucleotide may encode an antigen that induces an immune response against an infectious pathogen or against tumor cells (Restifo, N. P. et al., Folia Biol. 40:74-88 (1994); Ulmer, J. B. et al., Ann. NY Acad. Sci. 772:117-125 (1995); Horton, H. M. et al., Proc. Natl. Acad. Sci. USA 96:1553-1558 (1999); Yagi, K. et al., Hum. Gene Ther. 10: 1975-1982 (1999)). The polynucleotide may encode an immunomodulatory polypeptide, e.g., a cytokine, that diminishes an immune response against self antigens or modifies the immune response to foreign antigens, allergens, or transplanted tissues (Qin, L. et al., Ann. Surg. 220:508-518 (1994); Dalesandro, J. et al., J. Thorac. Cardiovasc. Surg. 111: 416-421 (1996); Moffatt, M. and Cookson, W., Nat. Med. 2:515-516 (1996); Ragno, S. et al., Arth. and Rheum. 40:277-283 (1997); Dow, S. W. et al., Hum. Gene Ther. 10:1905-1914 (1999); Piccirillo, C. A. et al., J. Immunol. 161:3950-3956 (1998); Piccirillo, C. A. and Prud'homme, G. J., Hum. Gene Ther. 10: 915-1922 (1999)). For therapeutic polypeptide delivery, the polynucleotide may encode, for example, an angiogenic protein, hormone, growth factor, or enzyme (Levy, M. Y. et al., Gene Ther. 3:201-211 (1996); Tripathy, S. K. et al., Proc. Natl. Acad. Sci. USA 93:10876-10880 (1996); Tsurumi, Y. et al., Circulation 94:3281-3290 (1996); Novo, F. J. et al., Gene Ther. 4:488-492 (1997); Baumgartner, I. et al., Circulation 97:1114-1123 (1998); Mir, L. M. et al., Proc. Natl. Acad. Sci. USA 96:4262-4267 (1999)). For amelioration of genetic defects, the polynucleotide may encode normal copies of defective proteins such as dystrophin or cystic fibrosis transmembrane conductance regulator (Danko, I. et al., Hum. Mol. Genet. 2:2055-2061 (1993); Cheng, S. H. and Scheule, R. K., Adv. Drug Deliv. Rev. 30:173-184 (1998)). 
However, the efficiency of a polynucleotide uptake and expression, especially when the polynucleotide is not associated with infectious agents, is relatively low. For example, Doh, S. G. et al., Gene Ther. 4:648-663 (1997) report that the administration of plasmid DNA into mouse muscle results in the detectable transduction of an average of only 6%, i.e., about 234 out of approximately 4000 of the myofibers in the injected muscle. Also notable is that of the myofibers transfected, the actual number of transfected nuclei is presumed to be a small proportion.
Wheeler, C. J. et al., ibid., show that administration of plasmid DNA complexed with cationic lipid into a mouse lung results in the transduction of less than 1% of the lung cells.
Attempts have been made to increase the efficiency of in vivo polynucleotide administration into vertebrates using chemical agents or physical manipulations. Such chemical agents include cellular toxins such as bupivacaine, cardiotoxin or barium chloride (Wells, D. J., FEBS Letters 332:179-182 (1993); Vitadello, M. et al., Hum. Gene. Ther. 5:11-18 (1994); Danko, I., et al., Hum. Mol. Genet. 2:2055-2061 (1993); Fomsgaard, et al., Apmis 106:636-646 (1998); Fomsgaard, A., Immunol. Lett. 65:127-131 (1999)) which act to cause muscle damage followed by muscle regeneration by cell division which makes the cells more receptive to DNA entry (Thomason, D. B. and Booth, F. W., Am. J. Physiol. 258:C578-581 (1990)); polymers such as polyvinyl pyrolidone, polyvinyl alcohol, polyethyleneimine, polyamidomine, and polyethylene glycol-polyethyleneimine-transferrin complexes that coat the DNA and protect it from DNases and enhance plasmid DNA-based expression or immune responses (Mumper, R. J., et al., Pharm. Res. 13:701-709 (1996); Mumper R. J., et al., J. Cont. Rel. 52:191-203 (1998); Anwer, K., et al., Pharm. Res, 16:889-895 (1999); Boussif O., et al., Proc. Natl. Acad. Sci. USA 92:7297-7301 (1995); Orson F. M., et al., J. Immunol. 164:6313-6321 (2000); Turunen M. P., et al., Gene Ther. 6:6-11 (1999); Shi N.Y., et al., Proc. Natl. Acad. Sci. USA. 97:7567-7572 (2000)); particles that interact with the DNA and act as carriers and enhance DNA expression such as narrospheres, microspheres, dendrimers, collagen and polylactide co-glycolides (Leong K. W., et al., J. Controlled Release 53:183-193 (1998); Baranov A., et al., Gene Ther. 6:1406-1414 (1999); Lunsford L., et al., J. Drug Targeting 8:39-50 (2000); Bertling W. M., et al., Biotechnol. Appl. Biochem. 13:390-405 (1991)), bulking agents such as sucrose that are injected before DNA injection to help expand the spaces between muscle cells and therefore allow better distribution of the subsequently injected DNA (Davis, H. L. et al., Hum. Gene Ther. 4:151-159 (1993)); detergents such as sodium glycocholate, sodium deoxycholate, beta-cyclodextrin and Exosurf® surfactant that may increase or decrease DNA expression (Freeman D. J. and Niven R. W., Pharm. Res. 13:202-209 (1996); Raczka E., et al. Gene Ther. 5:1333-1339 (1998)), cationic or non-cationic lipids that may facilitate DNA entry into lipid bilayers of cells (Liu Y., et al., Nat. Biotechnol. 15:167-173 (1997); Eastman S. J., et al. Hum. Gene Ther. 8:313-322 (1997); Simoes, S., et al., Biochim. Biophys. Acta Biomembranes 1463:459-469 (2000); Thierry, A. R., et al., Gene Ther. 4:226-237 (1997); Floch V., et al. Biochim. Biophys. Acta Biomembranes 1464:95-103 (2000); Egilmez N. K., et al. Biochem. Biophys. Res. Commun. 221:169-173 (1996)), DNA binding agents such as histones or intercalaters that protect the DNA from DNases (Manthorpe, M., et al., Hum. Gene Ther. 4:419-431 (1993); Wolff, J. A., Neuromuscul. Disord. 7:314-318 (1997): WO 99/31262) or agents that enhance plasmid DNA transcription such as histone deacetylase inhibitor FR901228 or 8-Bromo-cyclic AMP (Yamano, T., et al., Mol. Ther. 6:574-580 (2000); Aria H., et al. Gene Ther. 7:694-702 (2000)). Physical manipulations include removal of nerves that control muscle contraction (Wolff, J. A., et al., BioTechniques 11:575-585 (1991)); electroporation that electrically opens muscle cell pores allowing more DNA entry (Aihara, H. and Miyazaki, J., Nature Biotechnol. 16:867-870 (1998); Mir, L. M., et al., C R Acad Sci. III 321:893-899 (1998), Mir, L. M., et al., Proc. Natl. Acad. Sci, USA 96:4262-4267 (1999); Mathiesen, I., Gene Ther. 6:508-514 (1999); Rizzuto, G., et al., Proc. Natl. Acad. Sci. USA 96:6417-6422 (1999)); use of intravascular pressure (Budker, V., et al., Gene Ther. 5:272-276 (1998)); use of sutures coated with plasmid DNA (Labhasetwar, V., et al,, J. Pharm. Sci. 87:1347-1350 (1998); Qin, Y., et al., Life Sci. 65:2193-2203 (1999)); use of sponges soaked with DNA as intramuscular depots to prolong DNA delivery (Wolff, J. A., et al. (1991), Ibid.); use of special needle-based injection methods (Levy, M. Y., et al., Gene Ther. 3:201-211 (1996); Doh, S. G., et al. (1997), Ibid.); and of needleless-injectors that propel the DNA into cells (Gramzinski, R. A., et al., Molec. Med. 4:109-118 (1998); Smith, B. F., et al., Gene Ther. 5:865-868 (1998); Anwer, K., et al. (1999) Ibid.). In addition, Wolff, J. A., et al. (1991) Ibid. and Manthorpe, M., et al., (1993) Ibid. refer to conditions affecting direct gene transfer into rodent muscle in vivo.
WO99/64615 identifies the use of products and methods useful for delivering formulated nucleic acid molecules using electrical pulse voltage delivery. Examples include the formulation of plasmid DNA in a saline solution containing agents that promote better delivery of the plasmid DNA into cells in vivo when the formulation is delivered with an electrical pulse. Electrical pulse delivery often comprises electroporation where an electrical pulse is delivered to a tissue that is previously injected with a drug. Electroporation of a tissue causes transient interruption of cell membranes allowing more drug to enter the cell through the interruptions or “pores.” The agents in the saline DNA solution that promote delivery of the DNA into electroporated tissues include propylene glycols, polyethylene glycols, poloxamers (block copolymers of propylene oxide and ethylene oxide), or cationic lipids. The WO99/64615 publication claims that the way that these agents enhance delivery of the DNA into cells is by either protecting the DNA from degradation by DNases or by condensing the DNA into a smaller form, or both.
U.S. Pat. No. 5,470,568 describes the use of surface active copolymers to enhance repair of permeablized cells, treat tissue damage, and to increase the efficiency of incorporation of exogenous molecules, e.g., DNA into cells in vitro. The '568 patent describes the use of poloxamers for these purposes, either with or without the use of a high energy phosphate compound, for example, ATP or phosphocreatine.
Many of these attempts to enhance tissue transduction have used agents that destroy muscle (bupivacaine, barium chloride) and actually lower expression (Norman, J. et al., Methods in Molec. Med. 29:185-196 (1999)); have to be pre-injected before the DNA (sucrose); are expensive organic polymers (polyvinyl pyrollidine), mutagens (intercalaters), antigenic proteins (histones) or devices that destroy muscle tissue (needleless or needle-free injectors); or need to be inserted surgically (sutures, sponges, intravascular pressure). Furthermore, most of these methods may be expensive and not suitable or practical for human use.
On the other hand, little attention has been given to the use of alternative salt solutions and/or auxiliary agents in the pharmaceutical formulation as a way of enhancing the efficiency of a polynucleotide-based polypeptide delivery. Investigators in this field routinely use normal saline or phosphate buffered saline (“PBS”: 0.9% (i.e., about 154 mM) NaCl and 10 mM Na-phosphate) solutions for polynucleotide delivery, e.g., by intramuscular injection, because they are physiologically isotonic, isoosmotic, stable, non-toxic, and also because they have been traditionally used for human intramuscular injections of other drugs. Sodium phosphate, in the absence of saline, has been used in humans for delivery of non-polynucleotide-based (e.g., small molecules) administered via the intramuscular or intravenous routes (See generally, Physician's Desk Reference. Medical Economics Co, Monyvale, N.J. (1998)).
Sodium or potassium phosphate have been reported to enhance Lipofectin™-mediated transfection of human osteosarcoma cells in vitro (Kariko, K., et al., Biochim Biophys Acta 1369:320-334 (1998)), and the use of RPMI cell culture medium buffered with NaHCO3/Na2HPO4 were reported to be the best medium for forming DNA/cationic lipid complexes in vitro. (Kichler, A., et al., Gene Ther. 5:855-860 (1998)). 
Poloxamers have been approved for human use for intramuscular, intravenous, intraventricular, oral and topical administration. For example, Poloxamer 188 has been used as an adjunct to primary percutaneous transluminal coronary angioplasty for acute myocardial infarction (O'Keefe et al, Am. J Cardiol., 78:747-750 (1996)). Poloxamer 188 (RheothRx) has also been used in a pilot study on acute painful episode of sickle cell disease (Adams-graves et al., Blood, 90:5:2041-2046 (1997)).
There remains a need in the art for a convenient and safe way of improving the effectiveness of in vivo polypeptide delivery via direct administration of a polynucleotide. Aqueous solutions of certain salts including sodium phosphate have been used in humans (i.e., intramuscular injection of various small molecule drugs), and detergents or surfactants as auxiliary agents are common additives in drugs administered into human tissues. However, the use of certain salts or auxiliary agents, or a combination thereof to improve the transduction, i.e., the entry into cells, and/or expression-enhancing efficiency of polynucleotides delivered in vivo is new.